Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: separation of growth factor and bradykinin pathways.
Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration [( Ca2+]i). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+]i response to these agonists was examined by four methods. Low concentrations of TPA (2 x 10(-10) M) had no effect on Ca2+ release due to EGF, TGR-alpha or bradykinin but resulted in a rapid return of [Ca2+]i to baseline levels for EGF or TGF-alpha. Addition of the PKC inhibitor staurosporine (1 and 10 nM) completely inhibited the action of TPA on EGF-induced [Ca2+]i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 microM TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-alpha. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF-alpha resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF-alpha in this cell type. A431 cells treated with higher concentrations of TPA (5 x 10(-8) M) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF-alpha but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF-alpha gave a single transient of [Ca2+]i, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-alpha) and bradykinin resulted in two [Ca2+]i transients equal in size to those obtained with a single agonist.(ABSTRACT TRUNCATED AT 400 WORDS)
Wheeler
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LA
Department of Biological Sciences, Discovery Research Allegan, Inc./Herbert Labs, Irvine, California 92715.
Goodrum
D D
DD
Sachs
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G
D000470:Alkaloids / Q000494:pharmacology; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D015759:Ionomycin / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011744:Pyrimidinones / Q000494:pharmacology; D015398:Signal Transduction; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D011744:Pyrimidinones; D013844:Thiazoles; D016211:Transforming Growth Factor alpha; D008345:Manganese; D015759:Ionomycin; D004815:Epidermal Growth Factor; C047505:R 59022; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium